Hyaluronan synthase gene and uses thereof

• US 20060013829A1
• Filed: 09/14/2005
• Published: 01/19/2006
• Est. Priority Date: 07/01/1994
• Status: Active Grant

• Current Assignee: Kshama Kumari, Paul Deangelis, Paul H. Weigel
• Original Assignee: Kshama Kumari, Paul Deangelis, Paul H. Weigel
• Inventors: Weigel, Paul H., Kumari, Kshama, DeAngelis, Paul
• Granted Patent: US 7,087,413 B2
• Time in Patent Office: Days
• US Class Current: 424/200.100
• CPC Class Codes: A61K 39/102 Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: Y10S 435/836 Bacillus licheniformis
• CPC Class Codes: Y10S 435/839 Bacillus subtilis
• CPC Class Codes: Y10S 435/848 Escherichia

Abstract

The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active Streptococcus equisimilis hyaluronate synthase (seHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product. Hyaluronate is also known as hyaluronic acid or hyaluronan.

Claims

1. A method of producing hyaluronic acid, comprising the steps of:

providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes at least two amino acid motifs selected from the group consisting of;

(SEQ ID NO;

11)(i)GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X)_3-7(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;

(SEQ ID NO;

12)(ii)(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)(I/V/C)(S/G)GPL(G/S)XY(K/R);

(SEQ /D NO;

13)(iii)(D/E)X(W/Y)(X)₂QXF(L/M)G(X)₂(C/V)(S/T)XGDDR(H/R/C)LTN;

and (SEQ /D NO;

14)(iv)(E/D)(T/S/V)P(X)₅(W/Y/F)(L/I)XQQ(T/N)RW(S/T/N)KS(Y/W/F)(F/C)RE,

with X being selected from the group consisting of any amino acid; and

wherein the at least two amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and

culturing the host cell under conditions appropriate for the production of hyaluronic acid.

2. The method of claim 1, further comprising the step of separating the hyaluronic acid from the host cell.

3. The method of claim 1, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

4. The method of claim 1, wherein the hyaluronan synthase gene is isolated from a microbial source.

5. A method of producing hyaluronic acid, comprising the steps of:

providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes at least three amino acid motifs selected from the group consisting of;

(SEQ ID NO;

11)(i)GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X)_3-7(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;

(SEQ ID NO;

12)(ii)(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)(I/V/C)(S/G)GPL(G/S)XY(K/R);

(SEQ ID NO;

13)(iii)(D/E)X(W/Y)(X)₂QXF(L/M)G(X)₂(C/V)(S/T)XGDDR(H/R/C)LTN;

and (SEQ ID NO;

14)(iv)(E/D)(T/S/V)P(X)₅(W/Y/F)(L/I)XQQ(T/N)RW(S/T/N)KS(Y/W/F)(F/C)RE,

with X being selected from the group consisting of any amino acid; and

wherein the at least three amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and

culturing the host cell under conditions appropriate for the production of hyaluronic acid.

6. The method of claim 5, further comprising the step of separating the hyaluronic acid from the host cell.

7. The method of claim 5, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

8. The method of claim 5, wherein the hyaluronan synthase gene is isolated from a microbial source.

9. A method of producing hyaluronic acid, comprising the steps of:

providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes the amino acid motifs;

(SEQ ID NO;

     11)(i)GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X)_3-7(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;

(SEQ ID NO;

12)(ii)(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)(I/V/C)(S/G)GPL(G/S)XY(K/R);

(SEQ ID NO;

13)(iii)(D/E)X(W/Y)(X)₂QXF(L/M)G(X)₂(C/V)(S/T)XGDDR(H/R/C)LTN;

and (SEQ ID NO;

14)(iv)(E/D)(T/S/V)P(X)₅(W/Y/F)(L/I)XQQ(T/N)RW(S/T/N)KS(Y/W/F)(F/C)RE,

with X being selected from the group consisting of any amino acid; and

wherein the amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and

culturing the host cell under conditions appropriate for the production of hyaluronic acid.

10. The method of claim 9, further comprising the step of separating the hyaluronic acid from the host cell.

11. The method of claim 9, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

12. The method of claim 9, wherein the hyaluronan synthase gene is isolated from a microbial source.

13. A method of producing hyaluronic acid, comprising the steps of:

providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes at least two amino acid motifs selected from the group consisting of;

(SEQ ID NO;

15)(i)GKR(X)₆F(X)_11-15DSDT(X)_8E;

(SEQ ID NO;

16)(ii)RY(X)₂AF(X)₂ER(X)₂QS(X)₉GPL(X)₂Y;

(SEQ ID NO;

17)(iii)QXFXG(X)₅GDDRXLTN;

and (SEQ ID NO;

18)(iv)P(X)₈QQXRWXKS(X)₂RE,

with X being selected from the group consisting of any amino acid; and

wherein the at least two amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and

culturing the host cell under conditions appropriate for the production of hyaluronic acid.

14. The method of claim 13, further comprising the step of separating the hyaluronic acid from the host cell.

15. The method of claim 13, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

16. The method of claim 13, wherein the hyaluronan synthase gene is isolated from a microbial source.

17. A method of producing hyaluronic acid, comprising the steps of:

providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes at least three amino acid motifs selected from the group consisting of;

(SEQ ID NO;

15)(i)GKR(X)₆F(X)_11-15DSDT(X)₈E;

(SEQ ID NO;

     16)(ii)RY(X)₂AF(X)₂ER(X)₂QS(X)₉GPL(X)₂Y;

(SEQ ID NO;

17)(iii)QXFXG(X)₅GDDRXLTN;

and (SEQ ID NO;

18)(iv)P(X)₈QQXRWXKS(X)₂RE,

with X being selected from the group consisting of any amino acid; and

wherein the at least three amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and

culturing the host cell under conditions appropriate for the production of hyaluronic acid.

18. The method of claim 17, further comprising the step of separating the hyaluronic acid from the host cell.

19. The method of claim 17, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

20. The method of claim 17, wherein the hyaluronan synthase gene is isolated from a microbial source.

21. A method of producing hyaluronic acid, comprising the steps of:

providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes the amino acid motifs;

(SEQ ID NO;

15)(i)GKR(X)₆F(X)_11-15DSDT(X)₈E;

(SEQ ID NO;

16)(ii)RY(X)₂AF(X)₂ER(X)₂QS(X)₉GPL(X)₂Y;

(SEQ ID NO;

17)(iii)QXFXG(X)₅GDDRXLTN;

and (SEQ ID NO;

18)(iv)P(X)₈QQXRWXKS(X)₂RE,

with X being selected from the group consisting of any amino acid; and

wherein the amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and

culturing the host cell under conditions appropriate for the production of hyaluronic acid.

22. The method of claim 21, further comprising the step of separating the hyaluronic acid from the host cell.

23. The method of claim 21, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

24. The method of claim 21, wherein the hyaluronan synthase gene is isolated from a microbial source.